Easytag express35s protein labeling mix 35s cys met

Two million cells were plated in 6 cm plates and allowed to grow for 40 hr. Cells were pulse labeled for 1 hr with 120 μCi of EasyTag Express³⁵S Protein Labeling Mix [³⁵S]-Cys/Met (PerkinElmer; Waltham, MA). Immediately after the radioactive pulse, cells were washed with PBS and either lysed in MNT with a protease inhibitor cocktail (Halt protease and phosphatase inhibitor single-use cocktail, Thermo Fisher) and 20 mM NEM, or chased for indicated time using regular growth media. Where indicated, cells were treated with 500 μM DNJ for 30 min prior to [³⁵S]-Cys/Met labeling and through the chase. Cell lysates were shaken for 5 min at 4°C, centrifuged at 17,000 g for 5 min at 4°C, and the supernatants were collected. Samples were pre-cleared with a 20 μl bed volume of protein-A sepharose beads (GE Healthcare) by end-over-end rotation for 1 hr at 4°C. The supernatants were collected and incubated with a 30 μl bed volume of protein-A-sepharose beads and 1.5 μl of α-IGF-1 receptor β (D23H3) XP (Cell Signaling) per sample. Samples were washed with MNT without protease inhibitors or NEM and eluted in sample buffer. Samples were then resolved on a 9% reducing SDS-PAGE, imaged using a GE Typhoon FLA 9500 phosphorimager (GE Healthcare), and quantified using ImageJ.

Samples were processed as previously described (13 (link)). Cells were grown for 24 h before transfection with TTC17 S-tag cDNA or left untreated. Twenty-four hours posttransfection the cells were pulse labeled for 1 h with EasyTag Express³⁵S Protein Labeling Mix [³⁵S]-Cys/Met (PerkinElmer). After 1 h of pulse labeling, the cells were washed 2× with PBS on ice. Cells were resuspended in cold homogenization buffer (10 mM Hepes, pH 7.4, 10 mM KCl, 1.5 mM MgCl, 5 mM sodium EDTA, 5 mM sodium EGTA, and 0.25 M sucrose) and passed through a 25-gauge needle 20×. All subsequent steps were conducted at 4 °C. The homogenate was centrifuged at 1000g for 10 min to pellet the nuclear fraction. The remaining supernatant was centrifuged at 45,000 rpm in a Beckman rotor (TLA 120.2) for 10 min to separate the cytosol (supernatant) from the endomembrane compartments (microsomes). The microsomes were resuspended in homogenization buffer containing 0.1 M NaCl and 10 μg trypsin, with or without Triton X-100 added to a final concentration of 0.1%. After incubation at 27 °C for 15 min, the reaction was quenched with 100 μg soybean trypsin inhibitor. Reducing sample buffer was added and analyzed via autoradiography.